Molecular separation column and use thereof

ABSTRACT

A molecular separation chromotography column for effecting the differential distribution, between two phases, of the components of a sample flowing therethrough. The column contains a substantially homogeneous solid stationary phase which comprises a porous matrix of fiber having particulate immobilized therein, wherein at least one of said fiber or particulate is effective for molecular separation. The column is characterized by a reduced pressure drop, increased axial dispersion, more uniform peak shapes and better separations at high sample loading.

RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 184,822 filedSept. 8, 1980, now abandoned, the entire disclosure of which isincorporated herein by reference.

This application is also related to the following copending application:

U.S. Ser. No. 276,982, filed June 24, 1981 entitled: "Process forPreparing a Zero Standard Serum" to Hou; and

U.S. Ser. No. 238,686, filed Feb. 27, 1981 entitled: "Tissue CultureMedium" to Cone et al, abandoned.

All of these aforementioned copending applications and the contentsthereof are not prior art with respect to the invention described andclaimed in this application.

BACKGROUND OF THE INVENTION

Numerous techniques exist for the molecular separation of the componentsof a given sample for either analysis purposes or for productpreparation purposes. One type of molecular separation embraces avariety of processes for effecting differential distribution of thesample components between two phases and such processes are generallyreferred to as chromatography. The differential distribution is achievedby an interchange between a moving phase, which can be a liquid or gas,and a stationary phase.

1. FIELD OF THE INVENTION

This invention relates to novel molecular separation columns, e.g.chromatography columns, and more particularly to a novel stationaryphase for use in such columns.

Chromatography is a general term applied to a wide variety of separationtechniques based upon the sample interchange between a moving phase,which can be a gas or liquid, and a stationary phase. When gas is themoving phase (or "mobile phase" as referred to in chromatographicterminology), the technique is termed gas chromatography and when liquidis the mobile phase, the technique is termed liquid chromatography.

The so-called "chromatographic adsorption method" of analysis wasoriginated by the Russian botanist, M. Tswett, Ber. Deut. Botan. Ges.,24, 316, 1906, who used it for separating components of plant pigments.Little notice of this work was taken until 1931 and up to 1940 theemphasis was on frontal and displacement analysis. The theory ofchromatography was originated by Wilson, J. N., J. Amer. Chem. Soc., 62,1583, in 1940. Although the important historical role of this work hasbeen largely neglected because the quantitative theory did not allow fordiffusion or nonequilibrium between the mobile and the stationaryphases, Wilson offered a sound qualitative description of nonequilibriumand its important place in chromatography. He also explained the role oflongitudinal diffusion. Although the large spreading effects arisingfrom low adsorption and desorption rates can be diminished by decreasingthe flow rates, this in turn gives rise to spreading effects due tolarge longitudinal diffusion effects.

Following Wilson's work the Nobel prize winning paper of A. J. D. Martinand R. L. M. Synge, Biochem. J., 35, 1358, 1941, appeared whichintroduced the plate theory of chromatography and revolutionized liquidchromatography. They also suggested using a gas as the mobile phase. Gaschromatography was first tried by A. T. James and A. J. P. Martin,Biochem. J., 50, 6979, in 1952. Since then, however, voluminousliterature has been published dealing with gas-liquid and gas-solidchromatography, and gas chromatography has evolved into a sophisticatedanalytical technique.

Initially, liquid chromatography was performed in large diameter glasscolumns under normal pressure. These conditions led to long analysistimes and a generally tedious procedure. However in recent years, withthe introduction of high pressure pumps, advances in both intrumentationand column packings have occurred so rapidly that it has becomedifficult to keep pace with the amount of literature that is beingpublished, and liquid chromatography is rapidly gaining ground on gaschromatography in becoming of equal stature.

Separations can be classified into either analytical or preparativedepending on the objective. In analytical separations, the objective ishigh resolution separation and identification and quantification of thevarious components of a sample mixture. In preparative chromatography,on the other hand, the objective is the isolation of pure quantities ofthe desired constituents in the sample. Liquid chromatography isadvantageous over gas chromatography in that the former can be both anexcellent analytical as well as a preparative technique. Gaschromatography is very limited in its application as a preparative toolbecause of the very small sample sizes. In liquid chromatography, on theother hand, milligram to gram quantities of preparative separations arenot uncommon depending on the chromatographic column diameter and theamount of stationary phase.

The collection of chromatographic techniques can be classified inseveral ways and the most fundamental is based on naming the types ofphases used. Liquid absorption chromatography is used extensively fororganic and biochemical analysis but is limited because there are only afew suitable adsorbents. The distribution coefficient of adsorptionoften depends on total concentration and this behavior often results inincomplete separations. Gas-solid chromatography has generally sufferedfrom the same defects as liquid adsorption chromatography. Ion exchangechromatography is a special field of liquid-solid chromatography and isspecifically applicable to ionic species. Affinity chromatography isbased on the attraction (affinity) of a ligand bonded to the solidstationary phase for a given component of the sample. Liquid-liquid orpartition chromatography involves the use of a thin layer of liquid heldin place on the surface of a porous inert solid as the stationary phase.Paper chromatography is a special field of liquid-liquid chromatographyin which the stationary liquid is a film of water adsorbed on a papermat and thin layer chromatography is similar to paper chromatographyexcept that the paper is replaced by a glass or plastic plate coatedwith a thin layer of alumina, silica gel or other powdered material.

Column efficiency is generally measured in terms of H, sometimesreferred to HETP (height equivalent to a theoretical plate), which isthe column length divided by the total number of theoretical plates (n)contained in that length. H is generally considered to be a summation ofthree contributions, i.e. the contribution from non-equal paths (eddydiffusion), the contribution from diffusion along the column(longitudinal diffusion) and the contribution from non-equilibrium (masstransfer). The eddy diffusion is directly proportional to the diameterof the particles constituting the stationary phase. The less homogeneousthe structure, the larger is the contribution from non-equal paths.Conventional chromatographic theory thus predicts that finer packinggeometries will have decreased diffusional boundary layers, i.e. shorterpaths for material transport to the solid surface which will result inincreased efficiency. Current chromatographic theory, and thereforecurrent practice, leads to the use of very fine, homogeneous sphericalpacking. A complicating factor, however, is that the resistance of thecolumn to fluid flow, i.e. the pressure drop across the column, isinversely proportional to the square of the diameter of the particle.Therefore, halving the particle diameter will increase the pressure dropby a factor of 4.

Additionally, as known in the art, gel substrates are unable towithstand significant pressure drops and/or low flow rates.

2. PRIOR ART

The use of adsorbents or particulates carried by fibers or paper hasbeen used in the filter art for some time, see, for example, thefollowing U.S. Pat. Nos.

2,143,044 to Wicks et al;

2,746,608 to Briggs;

3,238,056 to Pall et al;

3,253,978 to Bodendorf et al;

3,591,010 to Pall et al;

4,007,113 to Ostreicher;

4,160,056 to Samejima; and

4,238,334 to Halbfoster.

Carbon has been loaded on a sheet having particles homogeneouslydistributed and firmly retained therein, as described in U.S. Pat. No.3,149,023 to Bodendorf et al. The Bodendorf et al sheets are used ascigarette filters, air filters, gas filters, wrappers for fruit andsubstances prone to discoloration or spoilage by gases in the atmospheredeodorizer layers in laminated sheet products for sanitary napkins, andfor surgical dressings for wounds, and the like.

Somewhat similar sheets, as described in the aforementioned patents,have been employed in paper and thin layer chromatography.

Malcolm, U.S. Pat. No. 3,647,684 teaches a thin layer chromatographymedium which takes the form of a self-supporting flexible sheetstructure having a major proportion of a chromatographic adsorbent suchas silicic acid uniformly and homogeneously dispersed with a minoramount of structurally stabilizing inorganic fibers such as glass fibersdisposed in a randomly oriented network of a cationic material such ascationic starch.

Leifield, U.S. Pat. No. 3,455,818 teaches sorbent sheets useful forchromatography carried out in the same general manner as conventionalthin layer or paper chromatography. The sheets are prepared bydispersing fine fibers of a non-cellulosic material such as fibrousglass together with a high proportion of the desired powdered sorbent ina suitable liquid medium which is flowed onto a porous support followedby removing the liquid. The sheets can be used in a column by rollingone or several of the sheets into a compact roll and inserting it into aglass tube or cylinder such that the interface between sheet surfaces isparallel to and in the path of the mobile phase flow.

Fibers or filament type packings for molecular separation columns havebeen used, see for example, Miller et al, The Use Of Textile Yarns InSeparation Processes, Textile Research Journal, Jan. 1980, pp. 10 etseq.; Brown et al, Macroreticular Resin Columns.I. Model of Bend andFilament Packings, Separation Science and Technology, 15(a), pp.1533-1553 (1980); and Partridge, Nature, 1123-1125 (March 18, 1967).Other references which suggest the use of fibers for molecularseparation packings are:

U.S. Pat. No. 3,570,673 to Dutzetal;

U.S. Pat. No. 3,307,333 to Norem et al;

U.S. Pat. No. 4,169,790 to Pretorius et al; and

U.S. Pat. No. 4,070,287 to Wiegand.

It is generally accepted in the art that scaling up from laboratoryresults is difficult, particularly in chromatographic processes wheretheoretical models are unsatisfactory. The construction of commercialinstallations based upon knowledge gained from laboratory experiments inthis field has turned out to be a major problem. According to thoseskilled in the art, the use of large columns of resins, for exampleorganic gel columns, is not desirable because of compaction, poorseparation results and because of excessive dilution of the elutedcomponents, both of which factors make the process an uneconomical one.If a liquid is introduced evenly across the top of the column, a portionof the front thereof moves downwardly at a rate different from the rateof movement of the balance of the liquid, running obliquely, causing"tailing" and "finger formation" to occur in the bed. To avoid theseproblems, it is desirable that the front or leading edge of each liquidor eluent, supplied to the top of the column, move downwardly at auniform rate, the front remaining substantially in a narrow band lyingin a horizontal plane.

These prior art problems are discussed, for example, by Baddour in U.S.Pat. No. 3,250,058. Good separations are achieved using thin laboratorycolumns, but when attempts are made to repeat the separation on atechnical or commercial scale, using columns of 5 cm or more indiameter, it is found that "tailing" and "finger formation" occur in thecolumn, both of which causes dilution and poor separation results.Baddour attempts to overcome these problems by the introduction of anarrangement of transverse baffles within the column to induce lateralflow of the liquid flowing through the column. In addition, Baddourfinds it necessary to use these baffles in combination with lateralbaffles.

The idea of a forced vertical flow in large scale columns was furtherdeveloped by Lauer et al. in U.S. Pat. No. 3,539,505, who introducedunits for radial mixing into the column or divided the column intoseveral short sections as described in German Patent Application DOS No.2,036,525. Yet another approach to the problem is described in theGerman patent application DOS No. 2,224,794 and Japanese Pat. No.73-68752, according to which the column is saturated with the solutionwhich is to be separated. By means of the saturated columns andcountercurrent flow, the disturbances caused by density gradients in thecolumn are avoided.

The rather complicated methods which are described above make itpossible to conduct large scale chromatographic separation procedures ona commercial basis. However, these methods lead to complicated columnstructures and to methods which are difficult to accomplish on acommercial scale. Where there are built-in structures within the column,substantial problems occur, for example, when the resin is backwashed.Backwashing is required in these procedures after a certain number ofcycles because mechanical impurities from the feed or eluent accumulateon the resin bed so that the performance of the column graduallydecreases in efficiency. It is obvious that built-in structures in thecolumn are a nuisance in such situations. The ideas of saturated resinbeds and countercurrent flow also lead to complicated structures asdescribed in the German Patent Application DOS No. 2,224,794 or to acomplicated procedure of operating the system.

Huber describes another approach in his U.S. Pat. No. 3,856,681 whereelongated rod-like elements were arranged parallel to the axis of thecolumn but those elements produced unsymmetrical column cross-sectionscausing difficult column packing and uneven fluid flow and also limitedoverall productive output of the column.

Huber, in his U.S. Pat. No. 3,856,681 attempts to obtain uniform flowacross a preparative or production chromatography column through the useof a plurality of layers of chromatographic media arranged adjacent toeach other, with the thickness dimension of the layers extendingsubstantially perpendicular to the primary fluid flow axis andpreferably spaced laterally from each other by relatively inertpartitioning means interposed between the layers. If desired, relativelylarge particles of chromatographic media or relatively inert materialcan be uniformly distributed through the chromatographic media layer toreduce the overall pressure drop through the final column. The surfaceof the chromatographic medium is parallel to and in the same directionas the mobile phase fluid flow.

McDonald et al in U.S. Pat. No. 4,211,656 describes a cartridge whichtriaxially compresses the particulate packing material to assure evenflow through the column.

It has now been discovered that a column in which a mobile phase flowsthrough a solid stationary phase can be constructed in directcontradiction to conventional chromatography packing theory if thestationary phase "system" is, broadly, a body of particulate immobilizedin a porous matrix of fiber. This new stationary phase has the advantageof both low pressure drop and low diffusion resistance making itparticularly suitable for commercial scale separations, particularlyliquid separations. Baffle arrangements are unnecessary. As a result, itis possible to construct stable, high flow separation columns of highcapacity and shorter run times which have good pressure response,freedom from channeling or fluid bypass, ease of regeneration toreproducible reuse, and the capacity to be shipped under ambientconditions or stored indefinitely. Additionally, the edges of the newstationary phase cooperate with the interior wall of the separationcolumn to form a substantially fluid tight seal therewith, thuspreventing channeling near the walls.

SUMMARY OF THE INVENTION

In accordance with the present invention, a molecular separation columnis provided for effecting differential distribution, between two phases,of the components of a sample flowing therethrough, said columncontaining a solid stationary phase which comprises a porous matrix offiber having particulate immobilized therein, at least one of said fiberor particulate being effective for molecular separation, preferably theparticulate, the matrix being substantially homogeneous with respect toeach component. When used in liquid-solid flow-through molecularseparations, there is a reduced pressure drop and diffusional resistanceso that the columns can be used for commercial scale liquid separationsin addition to analytical separations.

A method is also provided for effecting molecular separations by the useof such columns as well as providing for a solid phase for use in suchcolumns.

The columns of the present invention, when compared to conventionalcolumns containing similar particulate exhibit lower pressure drops; areless sensitive to high pressures (for example, pressures of 154 kg/cm²in a 10 mm diameter column do not effect column performance); exhibitmore axial dispersion (conventional columns exhibit greater dispersionof the separated components due to mass transfer resistance); exhibitbetter separation at high sample loadings; are less sensitive to flowrates; and exhibit more uniform peak shapes.

The solid stationary phase of the present invention has advantages incommercial scale chromatographic separations, particularly for highvolume, high molecular weight separations. Experiments have shown thatthe porous matrix provides greater eddy diffusion than particulate aloneand at 70% particulate, has a lower diffusional resistance, presumablydue to improved flow distribution in the more open matrix. Therelatively high eddy diffusion and low diffusion resistance suggest thatthe porous matrix has two unique features for chromatographicseparations--improved separations for components with low diffusioncoefficients, and more uniform peak shapes.

Conventional chromatographic theory is quite successful for modelingseparations with linear adsorption isotherms. Many separations areapparently linear at low sample concentrations but the separations ofcommercial interest are often relatively high concentrations andnon-linear. The efficiency of the stationary phase of the presentinvention obtained at high sample concentrations indicates that it iseffective for commercial separations.

OBJECTS OF THE INVENTION

An object of the present invention is to provide novel molecularseparation solid phase media and columns containing such media.

Another object is to provide novel media which have the characteristicsof low pressure drop and low diffusion resistance thereby making itparticularly suitable for commercial scale separation, particularly inliquid separations.

A further object is to provide stable, high flow separation columns ofgood capacity and shorter run times which have good pressure response,freedom from channeling or fluid bypass, ease of regeneration to areproducible result and the capacity to be shipped under ambientconditions or stored indefinitely.

Yet another object is to provide new solid stationary phase media whichcooperates with the interior wall of a separation column to form asubstantially fluid-tight seal and thereby prevent channeling near thewalls.

These and other objects of the invention will become apparent to thoseskilled in the art from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional elevational view of an embodiment of a packedmolecular separation column in accordance with the present invention;

FIG. 2 is a block diagram illustrating the use of the molecularseparation column of FIG. 1 in accordance with the present invention;

FIG. 3 is a block diagram of a second embodiment illustrating the use ofa molecular separation column in accordance with the present invention;

FIGS. 4A and 4B show simple fiber/fiber packings;

FIGS. 5 and 6 are chromatograms obtained in the separations of Examples1 and 2;

FIG. 7 is a plot of column efficiency as a function of linear velocityobtained from Example 1;

FIG. 8 is a plot showing the dependance of the factor (k') on linearvelocity obtained from Example 1; and

FIGS. 9 and 10 set forth van Deemter plots (sorptive efficiency) andcapacity data for a stationary phase of the present invention obtainedfrom Example 4.

DESCRIPTION OF THE INVENTION

As used throughout this specification, the term "molecular separation"means the separation of components of a given sample by taking advantageof differential size, physical characteristic(s) or chemicalcharacteristic(s) of the various molecules contained within that sample.The term "column" encompasses any container, usually but not necessarilycylindrical in shape, having total depth of at least one centimeter andpreferably greater than two centimeters. The terms "homogeneous" or"substantially homogeneous" which are used in this specification torefer to the solid stationary phase means that the stationary phase isof a uniform substantially uniform structure and/or composition in aplane transverse to the flow of sample through a column.

FIG. 1 shows a preferred molecular separation column (10) for effectingdifferential distribution of a sample components between two phases inaccordance with the present invention. The column (10) is a hollowcylinder (11) of circular cross-section which can be fabricated from anysuitable material such as glass, steel, plexiglass and the likecontaining a number of discs of solid stationary phase elements (12).The edges (13) of the elements (12) form a fluid-tight seal with theinterior wall of cylinder (11). The fluid-tight seal can be achieved inseveral ways. In one embodiment, the dimensions of the elements (12) andthe interior of the cylinder (11) are such that the elements (12) areheld firmly in place by a friction fit such that a pre-load compressesthe elements. This requires very precise dimensional tolerances for boththe interior wall of cylinder (11) and the elements (12). The individualelements (12) are inserted in the cylinder (11) usually with somemechanical aids such as a push-rod or piston. In a preferred embodimentwhich is suitable when an aqueous mobile phase is being passed throughthe column, the elements (12) are hydrophillic and swell somewhat uponcontact with the mobile phase forming the required fluid-tight seal withthe interior wall of cylinder (11). In such case, the dimensionaltolerances of the interior surface of the cylinder and the elements (12)need not be as precise as in the case of a friction fit.

The column (10) includes an inlet cap (15) held in place by bolts (16)and an outlet cap (17) held in place with bolts (18). Inlet cap (15) ismaintained in spaced relationship with cylinder (11) by spacer elements.Gasket rings (19) and (20) maintain an air-tight seal of caps (15) and(17) with cylinder (11). Inlet cap (15) is provided with an inletorifice (21) for receiving liquid introduced into the column and inletdiffuser (22) for distributing the incoming liquid across the bore ofthe cylinder. Outlet cap (17) is provided with a support screen (23) toretain elements (12) within the column and an outlet orifice (24)through which the separated liquid is discharged to a sample detectorfor analysis.

FIG. 2 is illustrative in diagrammatic form of the use of the column ofFIG. 1. Suitable solvents, such as Fisher Scientific and MCBHigh-Pressure Liquid Chromatography (HPLC) grade solvent, can becirculated from a solvent reservoir (100) by a constant volume pump(101) which provides a flow of 1-12 ml/min. such as is available fromAMF Cuno Division. A pulse dampener (102), which can be a two meterlength of 0.16 cm o.d.×0.08 cm i.d. stainless steel tubing followed by atee union with a pressure gauge, interconnects the pump (101) with aValco six port injection valve (103) at the head of a column (104).Suitable detectors (106) for the effluent include a Pharmacia 254 UVdetector for efficiency studies and phenol separations and a Varian(Palo Alto, California) Vari-chrom UV Visible Spectral Photometer forcapacity studies and separations requiring wavelengths other than 254nm. A variable range strip chart recorder (106) can enable the use ofeither detection system.

FIG. 3 is illustrative in diagrammatic form of another embodiment of theuse of a column in accordance with the present invention. A solvent froma suitable reservoir (200) is circulated by one or more high pressurepumps (201) which can be connected in series or in parallel to a highpressure pulse dampener (202), a Bourdon-type pressure gauge (203) and avalve-loop injector (204) into a column (205) which is fitted with abypass circuit (206). The column effluent is passed to a 254 nm fixedwave length filter photometer (207), which is connected to a suitablerecorder (208), and the effluent is then passed to a waste reservoir(209).

The stationary phase comprises a porous matrix of fiber havingparticulate immobilized therein, wherein at least one of said fiber orparticulate is effective for molecular separation. The porous matrix issubstantially homogeneous with respect to each component thereof.Preferably the particulate is effective for molecular separation. Themolecular separation particulate should be contained in the stationaryphase at an effective amount to achieve the desired molecularseparation. Preferably the porous matrix is substantially inert anddimensionally stable. FIG. 1 illustrates the use of a plurality ofdisc-shaped elements but a single column element of equivalent lengthcan also be used if so desired.

The preferred particulates which can be used in the present inventioninclude all of those substances which can be provided in finely dividedform and exhibit chromatographic functionality, i.e. capable ofeffective molecular separations. Mixtures of such compositions may alsobe utilized. Exemplary of such particulates are silica, alumina,diatomaceous earth, perlite, clays such as vermiculite, carbon such asactivated carbon, modified polymer particulates such as ion exchangeresins, crystalline cellulose, molecular sieves, and the like, thesurfaces of which may be modified in conventional manner.

Such materials are commercially available under a variety of trademarkssuch as Biosila, Hi-Flosil, LiChroprep Si, Micropak Si, Nucleosil,Partisil, Porasil, Spherosil, Zorbax Sil, Corasil, Pallosil, Zipax,Bondapak, LiChrosorb, Hypersil, Zorbax, Perisorb, Fractosil, CorningPorous Glass, Dowex and Amberlite resins and the like.

Examples of references which describe particulates effective formolecular separations are the following:

U.S. Pat. No. 3,669,841 to Miller;

U.S. Pat. No. 3,722,181 to Kirkland et al;

U.S. Pat. No. 3,795,313 to Kirkland et al;

U.S. Pat. No. 3,983,299 to Regnier;

U.S. Pat. No. 4,029,583 to Chang;

U.S. Pat. No. 3,664,967 to Stehl;

U.S. Pat. No. 4,053,565 to Krekeler; and

U.S. Pat. No. 4,105,426 to Iher.

The entire disclosures of all of these references are incorporatedherein by reference.

The particle size of the particulate is not critical but influencessomewhat the flow rate at which the sample to be treated passes throughthe present columns. Usually, uniform particle sizes greater than about5 microns are preferred with about 10-100 microns constituting apractical operational range. The amount of the particulate can varywidely from about 10 weight percent up to 80 weight percent or more ofthe solid stationary phase. The optimum particulate concentration willvary depending on the molecular separation desired although, at present,the greater concentrations of particulate appear to be more desirable.

The porous matrix of fiber may be any matrix capable of immobilizing theparticulate contained therein, i.e. capable of preventing significantparticulate loss from the stationary phase and having a porosity whichenables the fluid to pass through the column. Preferably, the porousmatrix is comprised of a self-bonding matrix of fibers. Suitable fiberswhich may be used in the present invention include polyacrylonitrilefibers, nylon fibers, rayon fibers and polyvinyl chloride fibers,cellulose fibers, such as wood pulp and cotton, and cellulose acetate.The preferred stationary phase of this invention has a porous matrixcomprised of a self-bonding matrix of cellulose fibers.

In order to provide a matrix which is a coherent and a handleablestructure for commerce and industry, it is desirable that at least oneof the components which go into forming the porous matrix is a long,self-bonding structural fiber. Such fiber gives the stationary phasesufficient structural integrity in both the wet "as formed" conditionand in the final dried condition. Such a structure permits handling ofthe phase, and in particular sheets during processing and at the time ofits intended use. Such fibers are typically available in diameters inthe range of 6 to 60 micrometers. Wood pulp, for example, has fiberdiameters ranging from 15 to 25 micrometers, and fiber lengths of about0.85 to about 6.5 mm.

The preferred long self-bonding structural fibers are preferablyobtained from normally dimensioned cellulose pulp such as manila hemp,jute, caroa, sisal, bleached or unbleached kraft, kozu and the like,which typically has a Canadian Standard Freeness of +400 to +800 ml.These long self-bonding fibers will constitute greater than 50% of theporous matrix, by weight, preferably about 66-90% of the porous matrixand most preferably 75-83%.

When the amount of particulate immobilized in the porous matrix is low,i.e. less than about 50% by weight of the media, it is preferred thatthe porous matrix be formed of a self-bonding matrix of normal cellulosepulp having a Canadian Standard Freeness of +400 to +800 ml.

In the preferred embodiment of this invention it is desirable to have ahigh amount of particulate, i.e. greater than about 50% by weight of thestationary phase, immobilized in the porous matrix. It is thus highlydesirable to use the invention described in copending application U.S.Ser. No. 123,467 to Hou et al to maintain such high content ofparticulate in the stationary phase. The entire disclosure of thisapplication is incorporated herein by reference. Broadly, a minorportion of cellulose pulp refined to a Canadian standard freeness ofbetween about +100 and -600 ml is incorporated with a major portion ofthe normally dimensioned cellulose pulp (+400 to +800 ml). Inparticular, from about 1% to about 10% of the refined pulp and about 10%to about 30% of the normal cellulose pulp, by weight of the stationaryphase, is contained in the stationary phase, the remainder being theparticulate. Generally, the weight ratio of unrefined to highly refinedpulp will range from about 2:1 to about 10:1, preferably 3:1 to about5:1. Such a mixture of pulps permits the retention of fine particulatesup to about 80% or more by weight of the stationary phase.

The amount of particulate in the stationary phase may be as little as10% by weight of the solid phase up to about 80% by weight. Preferably,levels of about 50 to 80% by weight are employed.

Preferably, the sheets which form the stationary phase, are formed byvacuum-felting an aqueous slurry of such normal cellulose fibers, highlyrefined wood pulp and particulate. This forms a sheet having theparticulate immobilized in a porous matrix. The sheet shows a uniformhigh porosity, fine pore-size structure with excellent flowcharacteristics and is substantially homogeneous with respect to thefiber and particulate.

The sequence of adding the required components to water to form theaqueous slurry appears to be relatively unimportant provided that theslurry is subjected to controlled hydrodynamic shear forces during themixing process. Preferably, the refined pulp is added to a slurry of theunrefined pulp and then the particulate incorporated therein. The slurryis normally prepared at an about 4% consistency and then diluted withadditional water to the proper consistency required for vacuum-feltingsheet formation. This latter consistency will vary depending upon thetype of equipment used to form the sheet. Typically the slurry is castonto a foraminous surface, vacuum felted, and dried in the conventionalmanner. The flat, dimensionally stable sheet can be of any desiredthickness and is then cut to the appropriate dimensions for each type ofcolumn. Alternatively, the elements, usually in the form of discs toaccommodate a chromatographic column, can be produced by dry mixing andpressing. A column of the stationary phase can also be formed in situ,for example by a slurry packing technique.

Chemical binders and additives may be used in forming the solidstationary phase of this invention. However, there should not be anychemical treatment detrimental to molecular separation.

A preferred molecular separation column in accordance with the inventioncomprises a plurality of elements, i.e. cut discs or pads, packed intothe column housing which is usually in the shape of a cylinder with avery precise internal diameter. The discs are cut to the same diameteras the cylinder and stacked in the cylinder to an appropriate height.The disc and cylinder should preferably be in interference fit such thatthe disc can be readily pushed into the cylinder to any requisite depthbut should not fall under gravitational force. After the column ispacked dry, a high pressure pump can be used to pump solvent through theelements stacked in the column. Preferably, the elements swell to form asubstantially bypass-free edge seal to the cylinder wall. The solventfront is very even with little or no skewing. Because the individualelements are dimensionally stable, the column is not sensitive toorientation or handling which is a common and major problem with otherchromatographic media, particularly organic gel media.

In general, the flow rates attainable with the molecular separationcolumns of this invention are substantially higher than those obtainablewith conventional gel or packed particle columns. The present columnsalso have excellent capacity since the diameter of the column is almostunrestricted compared to conventional gel or particle columns.

The molecular separation columns of this invention may be used for thewell-known molecular separations usually performed with conventionalcolumns.

Additionally the columns of the present invention may be found useful inareas where conventional columns are impractical, see for example theaforementioned copending and related applications U.S. Ser. No. 278,982,"Process for Preparing a Zero Standard Serum" to Hou and U.S. Serial No.238,686 "Tissue Culture Medium" to Cone et al.

Typically chromatographic separations prior to the present inventionhave led to the development of very fine, spherical in many cases,homogeneous solid stationary phase materials. Conventional theoreticalanalysis of chromatographic separations led to the conclusions that forhigh resolutions, practical pressure drop levels and useable flow rates,analytical type columns needed extremely small, spherical and uniformparticles with very specific surface properties. The use ofcomparatively non-homogeneous particles, i.e. fiber and particulate, ofwide size distribution, distributed in a bimodal (two size) fibermatrix, as is the stationary phase of the present invention, is indirect contradiction of everything known about chromatographicseparations.

The variables for the stationary phase of this invention that should beconsidered are, inter alia:

(1) type of fiber or fibers used;

(2) aspect ratio (L/D) of each type of fiber (initial and afterrefining);

(3) volume percent of each component in mixture;

(4) type of particulate or particulate mixture used;

(5) diameter of particulate (if spherical ) or other measure of particlegeometry (aspect ratio, etc.);

(6) solids level (S/L used in disc formation);

(7) ratio (R) of particulate size (diameter) to fiber diameter;

(8) type of resin or binder used (if any);

(9) surface modification of particulate (or of fiber) used;

(10) type of solution used for slurry (water, alcohol, solvent) to formsheets;

(11) slurry additives (wetting agents, impurities, etc.); and

(12) felting conditions.

If one assumes that the stationary phase of this invention will bedesigned using a fiber matrix (e.g. refined pulp and highly refinedpulp) and surface active particulate or particulate mixture chemicallytreated or derivatized prior to use for specific surface properties,including or not including the fiber, if desired, and binder systems,then the following simplified discussion will assist one skilled in theart in the design of the stationary phase.

A stationary phase with a high bulking factor is desirable. An open orporous matrix with high compression strength is needed for high flowrate at low pressure drops. These properties are primarily determined bythe fiber matrix and must be consistent with holding the particulate inthe structure. Therefore, the optimum mixture will vary from particulateto particulate.

If one assumes for discussion sake that the fibers are rigid rods ofvarious L/D ratios and that the particulate additives are spherical innature, the problem of stationary phase design from a structuralstandpoint becomes one of understanding the packing of fibers withfibers and fibers with spheres. Simple fiber/fiber packings are shown inFIGS. 4A and 4B. The density of a simple fiber system is a function ofthe fiber L/D ratio, with low L/D ratios leading to higher densities orlower interstitial volumes. The bulk volumes as a function of L/D ratiofor various rigid fibers is known. The bulk volume is essentiallyindependent of the fiber material (low modulus flexible fibers will havea different effect). If fiber mixtures are considered, the problem issomewhat more complicated and the resulting packing efficiency isdetermined by the ratio of the fiber L/D ratios. For fibers of the samediameter, fibers short enough to fit into the interstitial volume of thelonger fibers will create a lower bulk density material. Certain otherfiber size ratios can increase or decrease the resulting bulk volume.

Uniform spheres, if allowed to pack gravitationally, will form hexagonalpacked layers. Table 1 shows the effect of sphere diameter on theoccupied volume, the interstitial volume, the surface area, and surfaceto interstitial volume ratio.

                  TABLE 1                                                         ______________________________________                                        HEXAGONAL PACKED SPHERES                                                      COLUMN 7.6 cm DIA., 30.5 cm LONG                                                                0.3 cm   0.15 cm                                                              SPHERES  SPHERES                                            ______________________________________                                        OCCUPIED VOLUME, cm.sup.3                                                                         871.5      836.5                                          INTERSTITIAL VOLUME V.sub.1 cm.sup.3                                                              517.9      552.3                                          SURFACE AREA (S), cm.sup.2                                                                        16588      31850                                          S/V.sub.1           78.5       145                                            ______________________________________                                    

As expected, the surface to interstitial volume increases dramaticallyfor smaller diameters. While the total interstitial volume increasesonly slightly. What is not shown in this table, however, is that whilethe interstitial volume increases only slightly, the path through theinterstitial volume becomes much more tortuous because each component inthe path becomes much smaller (leading to higher pressure drops forsmaller diameter particles). The ratio of interstitial volume to surfacearea is important since this is the factor which primarily determinesthe equilibrium distribution of material from the liquid phase to thesolid surfaces in a typical chromatographic column. This simple spherepacking picture indicates why uniform spheres of small diameter givemore useful columns as long as the pressure drop does not becomeexcessive. Typically, particle diameters from 5-10 um are common inliquid chromatography. If small spheres are mixed with large spheres thebulk volume is a function of their diameter and volume fractions.

If simple uniform fibers are packed with uniform spheres, the efficientsphere packing is abruptly interrupted and the bulking factor is afunction of fiber L/D ratio and the ratio R of sphere diameter to fiberdiameter and the volume fraction of each component. This is shown inTable 2 which indicates the large density differences that are possiblewith composite blends depending upon the size and amount of eachcomponent.

                  TABLE 2*                                                        ______________________________________                                        THEORETICAL % SOLID CONTENT                                                   FOR FIBER-SPHERE PACKING                                                      L/D                                                                           % Fiber                                                                       Loading                                                                              R        1.00   2.00 3.91 7.31 15.51                                                                              24.49                                                                              37.10                         ______________________________________                                        10     R = ∞                                                                            68.5   68.5 68.5 68.5 68.5 68.5 61.0                                 R = 0    64.1   64.1 64.1 64.1 64.1 64.1 64.1                          20     R = ∞                                                                            77.0   77.0 77.0 77.0 69.0 56.2 44.3                                 R = 0    66.7   66.7 66.7 66.7 66.7 66.7 66.7                          30     R = ∞                                                                            87.7   87.0 83.3 77.5 60.0 46.2 34.5                                 R = 0    69.5   69.5 69.5 69.5 69.5 68.5 45.5                          40     R = ∞                                                                            85.5   83.3 78.7 72.0 52.9 39.1 28.6                                 R = 0    72.5   72.5 72.5 72.5 72.5 51.3 34.2                          50     R = ∞                                                                            82.7   80.6 74.6 67.2 47.2 34.1 24.2                                 R = 0    76.4   76.4 76.4 76.4 62.5 41.2 27.5                          60     R = ∞                                                                            80.0   77.5 71.0 62.9 42.7 30.2 21.0                                 R = 0    80.0   80.0 80.0 80.0 51.8 34.2 23.0                          70     R = ∞                                                                            77.5   74.6 67.6 59.2 39.1 27.0 23.0                                 R = 0    84.0   84.0 84.0 72.5 44.5 29.4 19.7                          80     R = ∞                                                                            74.6   72.0 65.0 56.2 36.0 24.5 16.7                                 R = 0    88.5   84.0 74.6 63.3 39.0 25.8 17.5                          90     R = ∞                                                                            72.5   69.5 62.2 53.2 33.4 22.4 15.1                                 R = 0    78.7   74.6 66.2 56.2 34.5 23.0 15.4                          100%            70.5   67.1 59.5 50.3 31.1 20.7 13.8                          Fiber                                                                         100%            61.5   61.5 61.5 61.5 61.5 61.5 61.5                          Spheres                                                                       Theoret.        88.5   87.4 84.4 80.6 73.5 69.5 66.7                          Max.                                                                          ______________________________________                                         *Milewski, John V., "Identification of Maximum Packing Conditions in the      Bimodal Packing of Fibers and Spheres," 29th annual Reinforced Plastics       Composites Conference, February 1974                                     

If the mixture consists of fiber size mixtures (various L/D) and spheresize mixtures (bimodal, trimodal, etc.), the packing density becomes acomplicated functional relationship to find more optimum materials.Higher bulk volume leads to high flow rate but there is a trade off withcompression strength. Other particle shapes and other fiber modulus(i.e. non-rigid fibers) will add their own complexity.

If one starts with a column of packed uniform spheres and adds a smallamount of fiber, it takes very little fiber to create a large change inthe interstitial volume of the resulting mix. The ratio of interstitialvolume to surface area will increase as will the size of the individualvolumes contributing to the total interstitial volume. This will lead tohigher flow rates but at the expense of the distribution equilibriumresponsible for the component separation. The literature is not clear asto the relative magnitude of these effects. There may be a regime ofhigh interstitial volume consistent with good uniform distribution ofthe active particulate but at a ratio of interstitial volume element toparticle surface area that is still consistent with good mediaseparation properties. These structure changes will influence thedistance over which diffusion takes place, the degree of mixing and flowturbulence, the tortuosity of the liquid path and other properties.Therefore, optimum structures for each specific system is to an extentempirical.

Notwithstanding theoretical explanations of the manner of operation, thepresent columns are characterized by a substantially reduced pressuredrop and more uniform chromatographic peaks. The present columns areeminently suited for preparative chromatography as well as foranalytical chromatography.

In order to further illustrate the invention, various examples are givenbelow. It will be appreciated that in these examples, as throughout thisspecification and claims, all parts and percentages are by weight andall temperatures in degrees Celsius unless otherwise indicated.

EXAMPLE 1

Weyerhauser Coho bleached Kraft, mean diameter about 20 microns, meanlength about 0.16 cm, Canadian Standard Freeness +600 ml. was refined ina Black Claussen twin disc refiner to a Canadian Standard Freeness of-250 ml. The refined pulp, unrefined pulp and an unmodified silica gelabout 15 microns in diameter were added to water with strong agitationto form an aqueous slurry of about 4% consistency. The slurry wasdiluted to 2% consistency and then vacuum felted in an about 16 cmdiameter hand sheet apparatus using a 100 mesh screen. The sheet wasremoved from the apparatus and dried in a static oven at about 175° C.until a constant weight was achieved. The sheet contained 70% of thesilica particulates and 30% wood pulp fiber (24% unrefined pulp and 6%highly refined cellulose pulp). The particulate was Syloid (W. R. Grace)620 having a nominal average particle size of 15 microns, a surface areaof 320 M² /g and a pore volume of 1.1 ml/g.

Discs were prepared and packed into a 50 cm precision bore stainlesssteel tube having a 10 mm inner diameter using a 9.5 mm diameter wooddowel. The discs were individually packed by hand into the column withthe screen side down and efforts were made to compress each disc to thesame degree.

Examination of the silica by scanning electron microscopy revealed thepresence of silica particles ranging in size from 1-40 microns, afeature which is generally unsuitable for chromatographic use due tohigh pressure drop and non-peak symmetry produced by a packed bed ofheterogeneously sized particles. The entire particle size range remainedentrained in the cellulose matrix. Some silica was lost during thefelting process such that the final composite contained 64% particulate.It was observed that the silica particles appear to be bound together bythe small, highly refined pulp and such fibers may, in fact, bechemically bound to the silica surface at the points of contact. Thecompletely packed column contained 15.7 grams of the composite materialand a void volume of 32.5 ml, i.e. 83% of the total column volume.

A test mixture containing three components was prepared. The componentswere chosen to provide information on peak dispersion for non-retained(k'=0, toluene), slightly retained (k'=2-3, dibutylphthalate "DBP") andwell-retained (k'=5-6, dimethylphthalate "DMP") solutes. The mobilephase composition (0.2% isopropanol in heptane) was adjusted to give theappropriate retention for these components on the stationary phase. Thecolumn was tested at flow rates of 0.2-19.9 ml/min. and thechromatographic characteristics (pressure drop, capacity factor and H)were calculated and the results are set forth in Table 3 below. The highdegree of peak symmetry displayed by the composite column throughout theevaluation is illustrated by a representative chromatogram set forth inFIG. 5.

                  TABLE 3                                                         ______________________________________                                        Flow   Pressure Toluene     DBP     DMP                                       Rate   Drop     t.sub.m H         H          H                                (ml/min)                                                                             (kg/cm.sup.2)                                                                          (min)   (mm)  k'  (mm)  k'   (mm)                             ______________________________________                                        0.2    2.1      166.43  0.51  2.3 0.65  5.6  0.85                             0.5    4.2      65.46   0.57  2.1 0.79  5.2  0.85                             1.0    8.05     32.48   0.65  2.2 0.90  5.5  0.87                             2.0    15.75    16.83   0.71  2.1 0.97  5.4  0.98                             4.0    29.75    8.46    0.73  2.1 1.05  5.4  1.06                             6.0    44.8     5.69    0.79  2.1 1.12  5.5  1.09                             8.0    59.5     4.31    0.75  2.2 1.17  5.7  1.09                             9.0    72.8     3.48    0.79  2.5 1.40  6.0  1.06                             ______________________________________                                    

The increase in pressure drop across the column was found to be a linearfunction of the flow rate (linear velocity) at pressures up to 70kg/cm². While this is not surprising for particulate packed beds, itindicates that virtually no compression of the matrix occurred at thesepressures. The column was also subjected to a pressure drop of 154kg/cm² which corresponded to the maximum flow rate capability of thesystem (19.8 ml/minute). Subsequent reevaluation of the chromatographicparameters at a flow rate of 6 ml/minute gave values (H_(DBP) =1.53,H_(DMP) =1.17) only slightly poorer than those previously obtained(Table 3). A void of 2.5 mm was, however, noted at the inlet end of thecolumn when the fitting was removed.

The influence of flow rate on chromatographic efficiency for thestationary phase is shown in FIG. 7. The general shape of the curves issimilar to that obtained with packed columns of porous silica. Althoughthe initial H increase for conventional columns is greater than for theporous matrix of this invention, this is offset somewhat by a laterflattening of the curve at high flow rates. The efficiency of the columnof the present invention in the region of relatively constant Hcorresponds roughly to that obtained with 30-35 micron porous silicaparticles and essentially no variation of the capacity factor was seenat the flow rates examined as indicated in FIG. 8.

EXAMPLE 2

For comparison, a 25 cm×10 mm i.d. column was packed to contain 10.3grams of the same silica particulate by a modified balance densityslurry technique. This was approximately equivalent to the 10 grams ofsilica calculated to be contained in the 50 cm column described inExample 1. Peak symmetry obtained with this column was generally poorand indicated a rather heterogeneous packed bed. The packed particlesapparently shifted somewhat during later use and pressures approachingthe initial packing pressure (ca 350 kg/cm²) and the peak symmetryimproved to that shown in FIG. 6. The chromatographic parameterscalculated for the silica column are shown in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Flow   Pressure Toluene     DBP     DMP                                       Rate   Drop     t.sub.m H         H          H                                (ml/min)                                                                             (kg/cm.sup.2)                                                                          (min)   (mm)  k'  (mm)  k'   (mm)                             ______________________________________                                        1.0    32.55    16.85   0.10  5.4 1.37  14.2 0.17                             2.0    55.3     8.44    0.09  --  --    --   --                               6.0    161.35   2.93    0.11  9.1 0.49  20.0 0.33                             9.9    267.4    1.83    0.12  3.8 0.56   9.6 0.34                             12.0   329      1.54    0.14  4.1 1.08  10.6 0.42                             ______________________________________                                    

For further comparison, a composite containing 94% unrefined celluloseand 6% of the -250 ml Canadian Standard Freeness cellulose and no silicawas evaluated in the 50 cm column. The calculated parameters are givenin Table 5 below.

                  TABLE 5                                                         ______________________________________                                        Flow      Pressure      Toluene                                               Rate      Drop          t.sub.m H                                             (ml/min)  (kg/cm.sup.2) (min)   (mm)                                          ______________________________________                                        0.2       2.1           125.86  3.68                                          0.5       4.9           49.39   4.10                                          1.0       8.4           24.61   4.20                                          2.0       16.1          12.60   4.63                                          4.0       30.1          6.46    4.59                                          ______________________________________                                    

The pressure drop was nearly identical to that of the compositecontaining particulate at all flow rates examined but the peakdispersion (H) for the unretained solute (toluene) was much greater. Thephthalates were unretained on the porous cellulose matrix.

EXAMPLE 3

Chromatographic characteristics of additional solid stationary phasematerials of the present invention were determined and the results areset forth in the following Tables 6-9:

                  TABLE 6                                                         ______________________________________                                        STATIONARY PHASE CONTAINS NOMINALLY                                           50% SILOID 620, 7.5% - 250 ml CSF PULP                                        AND REMAINDER UNREFINED PULP                                                  Flow   Pressure Toluene     DBP     DMP                                       Rate   Drop     t.sub.m H         H          H                                (ml/min)                                                                             (kg/cm.sup.2)                                                                          (min)   (mm)  k'  (mm)  k'   (mm)                             ______________________________________                                        1.0    2.45     16.70   5.09  1.0 6.36  2.8  5.28                             2.0    3.5      8.27    5.76  1.5 7.15  3.9  5.92                             4.0    4.9      4.33    5.82  1.5 7.02  3.7  6.41                             6.0    7.35     2.95    5.53  1.2 6.23  2.8  6.58                             8.0    9.45     2.17    6.09  1.0 7.62  2.8  6.51                             9.9    11.55    1.73    6.42  1.0 7.46  2.8  6.67                             ______________________________________                                         25 cm × 10 mm ID column containing 6.8 g of stationary phase            material.                                                                     Void volume = 16.7 ml = 85% of total column volume.                      

                  TABLE 7                                                         ______________________________________                                        STATIONARY PHASE CONTAINED NOMINALLY                                          44% SILOID 620, 6% - 250 ml CSF PULP                                          AND REMAINDER UNREFINED PULP                                                  Flow   Pressure Toluene    DBP      DMP                                       Rate   Drop     t.sub.m H          H          H                               (ml/min)                                                                             (kg/cm.sup.2)                                                                          (mm)    k'   (mm)  k'   (mm)  k'                              ______________________________________                                        1.0    4.55     15.60   2.33 1.0   2.91 2.6   2.51                            2.0    7.35     7.87    2.49 1.1   3.27 3.0   2.72                            4.0    12.25    4.04    2.68 1.1   3.36 3.0   2.73                            6.0    18.2     2.76    2.66 1.1   3.45 3.0   2.87                            8.0    23.8     2.05    2.65 1.1   3.52 3.0   2.94                            9.9    29.4     1.65    2.66 1.1   3.01 3.0   2.97                            ______________________________________                                         25 cm × 10 mm ID column containing 8.9 g of stationary phase            material.                                                                     Void volume = 15.6 ml = 80% of total column volume.                      

                  TABLE 8                                                         ______________________________________                                        STATIONARY PHASE CONTAINED NOMINALLY                                          19% SILOID 620, 6% - 250 CSF PULP                                             AND REMAINDER UNREFINED PULP                                                  Flow   Pressure Toluene     DBP     DMP                                       Rate   Drop     t.sub.m H         H          H                                (ml/min)                                                                             (kg/cm.sup.2)                                                                          (min)   (mm)  k'  (mm)  k'   (mm)                             ______________________________________                                        1.0    3.85     14.76   0.54  0.7 1.16  1.9  0.81                             2.0    6.65     7.48    0.55  0.7 1.31  1.9  0.85                             4.0    10.85    3.78    0.59  0.7 1.49  1.9  0.91                             6.0    16.1     2.56    0.62  0.7 1.49  1.9  0.97                             8.0    21       1.89    0.64  0.7 1.59  2.0  0.99                             9.9    25.9     1.56    0.61  0.7 1.67  1.9  1.04                             ______________________________________                                         25 cm × 10 mm ID column containing 9.9 g of stationary phase            material.                                                                     Void column = 14.8 ml = 76% of total column volume.                      

                  TABLE 9                                                         ______________________________________                                        STATIONARY PHASE COMPOSITE CONTAINED                                          NOMINALLY 30% SILOID 620, 10% - 250 CSF PULP,                                 AND REMAINDER UNREFINED PULP                                                  Flow            Toluene     DBP     DMP                                       Rate   Pressure t.sub.m H         H          H                                (ml/min)                                                                             kg/cm.sup.2                                                                            (min)   (mm)  k'  (mm)  k'   (mm)                             ______________________________________                                        1.0    4.55     15.60   2.33  1.0 2.91  2.6  2.51                             2.0    7.35     7.87    2.49  1.1 3.27  3.0  2.72                             4.0    12.25    4.04    2.68  1.1 3.36  3.0  2.73                             6.0    18.20    2.76    2.66  1.1 3.45  3.0  2.87                             8.0    23.8     2.05    2.65  1.1 3.52  3.0  2.94                             9.9    29.4     1.65    2.66  1.1 3.01  3.0  2.97                             ______________________________________                                         25 cm × 10 mm ID column containing 8.9 g of stationary phase.           Void volume = 15.6 ml = 80% of total column volume.                      

EXAMPLE 4

Using the column of Example 1, and the arrangement shown in FIG. 3,separations of phenols and phthalates were effected. The efficiency andcapacity data obtained are set forth in FIGS. 9 and 10, respectively.

The phenols were separated using 1% isopropyl alcohol in chloroform as amobile phase which yielded a solvent strength greater than the required0.3 to elute phenols from silica. The flow rate was 3.2 ml/min. and thecomponents varied between 10 and 25 mg. The components eluted in theorder of benzene, o-chlorophenol, p-phenylphenol, phenol andp-nitrophenol. Resolution of the first two components was approximately90%, the third and fourth components approximately 80% and the finalcompound greater than 98%. Although the phenol and p-phenylphenolappeared as a single peak in the mixture chromatogram, the chromatogramfor the pure components indicated a retention difference of 30 mm at achart speed of 48.6 cm/hour. When the pure component chromatograms wereoverlaid, a resolution of 88% was observed. The most striking aspect ofthe van Deemter plot shown in FIG. 9 is the shape of the curve. Normallyfor liquid chromatography, H values are expected to decrease at low flowrates. It has also been reported in the literature that at very low flowrates, the H values can begin to decrease and then increase sharply. Ascan be seen in FIG. 9, as the flow rate decreases, the H valuesdecrease, increase sharply and finally decrease again, a type ofbehavior that has not previously been reported.

The second set of chromatograms were obtained with the separation ofdimethyl, diethyl, dibutyl and dioctyl phthalates. The solvent systemused was 7% chloroform in hexane, the flow rate was 3.8 ml/min. and thechart speed was 40.6 cm/hr. The compounds eluted in the order ofdecreasing molecular weight. The resolution for octyl and butylphthalate was 95% or better; the resolution of the latter two compoundswas 92-94%. The peaks in the mixture chromatogram represented 18-22 mgof each compound. FIG. 10 shows that the stationary phase of the presentinvention had at least a 100 mg capacity.

EXAMPLE 5

Following the procedures and using the columns described in Example 4,the column parameters for a 99% separation of the phthalates with a 3.15cm/min. flow rate were determined. With the stationary phase containingnominally 70% particulate, the separation time was 2.38 minutes, thepressure drop was about 9.4 kg/cm², the distance the mobile phasetraveled was 5.71 cm and the total quantity of particulate was 1.14grams. For the silica particulate column, the separation time was 1.08minutes, the pressure drop was about 20.6 kg/cm², the distance themobile phase traveled was 1.54 cm and the total amount of particulatewas 0.705 gram. Substantially longer columns are required with thestationary phase of the present invention for the same degree ofseparation produced by a 100% particulate column. The additional columnlength, however, is more than offset by low pressure drops for theseparation. In commercial separations, the pressure drop characteristicsof the separation media is extremely important since excessively highpressure drops can limit the throughput of the column requiringexpensive pumps and materials or place a restraint on column length andthus the number of theoretical plates available for a given separation.Since the stationary phase of the present invention has a substantiallylower pressure drop than the 100% particulate column, much longer columnlengths are possible.

EXAMPLE 6

This example illustrates the effect that the composition of thematerials used in producing the solid stationary phase has upon thechromatographic effectiveness of the resulting stationary phase.

Eight aqueous mixtures of silica gel, long cellulosic fibers, and highlyrefined pulp were prepared, and the mixture was agitated for uniformity.The aqueous suspensions were then poured into a filter mold, removingthe water and forming a composite material felt. Eight felts wereproduced according to the experimental design presented in Table 10,with specific composition of the materials charged into the aqueoussuspension presented in Table 11. The eight felts were then removed fromthe filter and dried. Plugs (10 mm in diameter) were cut from the driedcomposite felt and packed into 250 mm stainless steel columns withconventional preparative high performance liquid chromatographic endfittings. The chromatographic evaluations of the thus formed stationaryphases were carried out using a mobile phase of 0.2 percent2-propanol/heptane. The pressure drop and the dispersion characteristicsof the 10 mm i.d. columns were evaluated at flow rates of 0.2, 6, and19.8 ml per minute. The dispersion characteristics for the stationaryphases were determined using an unretained solute (toluene), a slightlyretained solute (dibutyl phthalate, "DBP") and a well retained solute(dimethyl phthalate, "DMP"). The experimental results of the preparationof the fiber particulate materials as well as the chromatographiceffectiveness of these materials are presented in Tables 12 through 15.The effects of each of the composition variables on each set of theexperimental results were then obtained with a conventional statisticalanalysis of the screening design. The effect of a variable was thedifference between the average experimental result when the independentvariable was positive and the average value of the experimental resultswhen the independent variable was negative. The variable levels chosenfor run 8 were arbitrarily defined as the negative levels of thosevariables in this design. The significance of the effects was estimatedby comparing the effects of the independent variables to the effectsobtained with the dummy variable. The effect of the dummy variable isdue to experimental error as well as variable interactions. Because ofthe strong interactions that are potentially present in this screeningdesign, the effects of particle size, the dispersity of the particlesize distribution, and the presence of mixtures in the particulate werealso included in the estimate of the effects due to experimental errorand variable interactions. This results in a conservative estimate ofthe probability for significance, and the significance of the effectscould be substantially greater than appears in the tables. The analysesof the effects of the variables are presented in Tables 17 through 38.

Each of the variables that were investigated were significant ininfluencing the chromatographic performance of the stationary phase.Some of the compositional variables improved some of the experimentalresults but resulted in decreased performance in other areas. A summaryof the effects of the composition of the stationary phase on some of themore important experimental results is provided in Table 38.

As ther particle size of the Partisil brand porous silica was decreasedfrom 10 microns to 5 microns, there was a greater weight loss of thecomposite material during formation, the drainage time was reduced, thepressure drop through the sheet was greater, and the height equivalentof a theoretical plate (H in mm) was lower. The product of the pressuredrop and height equivalent of the theoretical plate was increased. Theoverall resolution of the column was not significantly increased by thechange in particle size of the Partisil. When the runs based on Partisilwere compared with the runs based on Siloid 620 brand silica, thePartisil decreased the felt drainage time and the resolution of thecolumn. The height equivalent of theoretical plate as well as theproduct of the pressure drop and height equivalent of the theoreticalplate was greater for the columns based upon Partisil than the columnsbased upon Syloid 620. When Partisil was present as a mixture withSyloid 620, the mixture had both lower pressure drops and lower heightequivalent of theoretical plates in the composite material than wheneither pure Syloid 620 or pure Partisil was used as the particulate. Theresolution of the columns when mixtures of Partisil and Syloid 620 werepresent was improved while the product of the pressure drop and heightequivalent of the theoretical plate was reduced, the weight lossassociated with the composite material formation increased, the feltdrainage decreased, and the height equivalent of the theoretical platedecreased. There was little effect on the pressure drop, the resolution,and the product of the pressure drop and the height equivalent of thetheoretical plate. There was a modest effect of the amount of therefined pulp on the capacity factor for the dimethyl phthalate. Thefreeness of the pulp was a significant variable since the runs with thehigher freeness values i.e. more positive CSF had lower weight loss,lower drainage time in the felt formation, lower pressure drop, and poorresolution with increased height equivalent of a theoretical plate. Ingeneral, the product of the pressure drop and height equivalent oftheoretical plates was reduced as the pulp freeness increased, however.The amount of particulate in the composite material was a significantvariable since reducing the amount of particulate in the compositematerial reduced the weight loss, the pressure drop, and the product ofthe pressure drop and height equivalent of the theoretical plate.Reducing the amount of particulate produced composite materials withsignificantly lower capacity factors, lower resolution, and a greaterheight equivalent of theoretical plates.

An important variable to assess the performance of the compositematerials for chromatographic separations is the product of the pressuredrop and the height equivalent of a theoretical plate. This product iscomparable to the pressure drop per theoretical plate, and a lower valuecorresponds to a more efficient separation, since at a given flow rateless pressure drop is required to achieve the separation of atheoretical plate. The combination of variables present in run 2resulted in a significant improvement in the value of the product of apressure drop and height equivalent of a theoretical plate, when theproduct was compared to the product of runs 4, 5, 7, and 8, in whicheither pure Partisil or pure Syloid 620 was used as the particulate. Theproduct of the height equivalent of a theoretical plate and the pressuredrop was lower in each run where Partisil was mixed with Syloid 620 thanthe runs produced with pure Partisil or pure Syloid 620 as theparticulate in the composite materials.

                                      TABLE 10                                    __________________________________________________________________________    EXPERIMENTAL DESIGN FOR SCREENING DESIGN                                      Experimental Variables                                                             1               4    5    6                                                   Particle        Amount                                                                             Refined                                                                            Amount of.sup.1                                Run  size 2     3    of refined                                                                         Pulp particulate                                                                          7                                       number                                                                             (μ)                                                                             Silica                                                                              Mixture                                                                            pulp (%)                                                                           freeness                                                                           %      Dummy                                   __________________________________________________________________________    1    5    Partisil                                                                            Yes  6    -100 70     -                                       2    10   Partisil                                                                            Yes  4    -250 60     -                                       3    10   Syloid 620                                                                          Yes  4    -100 70     +                                       4    5    Syloid 620                                                                          No   4    -100 60     -                                       5    10   Partisil                                                                            No   6    -100 60     +                                       6    5    Syloid 620                                                                          Yes  6    -250 60     +                                       7    5    Partisil                                                                            No   4    -250 70     +                                       8    10   Syloid 620                                                                          No   6    -250 70     -                                       __________________________________________________________________________     .sup.1 Remainder of composite material is unrefined pulp, i.e. long           cellulosic fibers.                                                       

                  TABLE 11                                                        ______________________________________                                        COMPOSITION OF MATERIALS IN                                                   INITIAL SCREENING DESIGN                                                      Run             Partisil       Amount Refined                                 num-  Syloid 620          Particle                                                                             of refined                                                                           Pulp                                  ber   (weight %)                                                                              (weight %)                                                                              size, (μ)                                                                         pulp (%)                                                                             Freeness                              ______________________________________                                        1     35        35         5     6      -100                                  2     30        30        10     4      -250                                  3     35        35        10     4      -100                                  4     60        0         --     4      -100                                  5     0         60        10     6      -100                                  6     30        30         5     6      -250                                  7     0         70         5     4      -250                                  8     70        0         --     6      -250                                  ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        RESULTS OF THE SCREENING DESIGN I                                                  Weight    Felt     Ash of          Resolu-                                    loss during                                                                             formation                                                                              com-   Pressure tion                                       sheet     time     posite drop (kg/cm.sup.2                                                                      at 6                                  Run  formation (sec)    (%)    at 6 ml/min)                                                                           ml/min                                ______________________________________                                        1    11        28       67     16.5     4.32                                  2    11        35       49     7.7      3.92                                  3    16        16       57     11.6     4.59                                  4     9        31       51     14       3.23                                  5    11        11       50     13.7     2.91                                  6    15        25       49     21.7     4.46                                  7    29        13       52     32.2     3.73                                  8    12        70       66     25.2     3.73                                  ______________________________________                                    

                  TABLE 13                                                        ______________________________________                                        RESULTS OF THE SCREENING DESIGN II                                                 H*        H*       H*     H*       H*                                         toluene   toluene  toluene                                                                              DMP      DBP                                        (.2       (6       (20    (.2      (.2                                   Run  ml/min)   ml/min)  ml/min)                                                                              ml/min)  ml/min)                               ______________________________________                                        1    0.758     0.482    0.610  0.922    0.904                                 2    0.606     0.746    0.802  1.120    0.964                                 3    0.580     0.331    0.431  0.930    0.833                                 4    0.756     0.921    1.050  1.450    1.240                                 5    1.060     1.940    2.170  2.790    2.040                                 6    0.611     0.332    0.451  1.320    0.790                                 7    0.778     0.600    0.726  1.700    1.240                                 8    0.711     0.518    0.712  1.900    1.070                                 ______________________________________                                         *H in mm                                                                 

                  TABLE 14                                                        ______________________________________                                        RESULTS OF THE SCREENING DESIGN III                                                H DBP    k' DBD   H DMP  k' DMP H × pressure                            (6       (6       (6     (6     drop (kg/cm.sup.2 at                     Run  ml/min)  ml/min)  ml/min)                                                                              ml/min)                                                                              (6 ml/min)                               ______________________________________                                        1    0.911    1.62     1.36   4.48   22.4                                     2    1.046    1.36     1.45   3.79   11.1                                     3    0.813    1.71     1.21   4.68   14                                       4    1.453    1.43     2.00   3.75   28                                       5    4.190    1.25     3.69   4.57   50.4                                     6    0.802    1.57     1.20   4.25   26                                       7    1.420    1.73     2.09   5.07   67.3                                     8    1.176    1.96     1.51   4.86   38                                       ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        RESULTS OF THE SCREENING DESIGN IV                                                                                 HETP ×                                  H DBP    k' DBD   H DMP  k' DMP pressure                                      (20      (20      (20    (20    drop (kg/cm.sup.2 at                     Run  ml/min)  ml/min)  ml/min)                                                                              ml/min)                                                                              20 ml/min)                               ______________________________________                                        1    1.120    1.65     1.26   4.63   79.7                                     2    1.210    1.38     1.59   3.76   42.3                                     3    0.957    1.65     1.30   4.49   55                                       4    1.770    1.45     1.86   3.81   89.8                                     5    2.880    1.32     2.97   3.16   141.4                                    6    0.938    1.64     1.16   4.40   87.7                                     7    1.610    1.68     1.90   4.95   226.2                                    8    1.480    1.95     1.61   4.79   144.3                                    ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        RESULTS OF THE SCREENING DESIGN V                                                  DBP, ratio of k'                                                                           DMP, ratio of k'                                                                           Ratio of the                                        at .2 and 20 at .2 and 20 pressure drop                                  Run  ml/min       ml/min       at 20 and 6 ml/min                             ______________________________________                                        1    1.260        1.200        3.850                                          2    1.610        1.510        3.450                                          3    1.050        1.070        3.670                                          4    1.290        1.260        3.450                                          5    1.300        1.320        3.480                                          6    1.110        1.140        3.480                                          7    2.080        1.940        3.710                                          8    0.964        0.810        3.550                                          ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON WEIGHT LOSS DURING SHEET FORMATION                                         Variable       Effect   T-value    Probability                                ______________________________________                                        1.  Particle size  3.50     0.83     52.90                                    2.  Dispersity     2.50     0.59     39.20                                    3.  Mixture        -2.00    0.47     31.43                                    4.  Amount of pulp 4.00     0.95     58.77                                    5.  Pulp freeness  -5.00    1.18     68.70                                    6.  Amount of particulate                                                                        -5.50    1.30     72.83                                    7.  Dummy          7.00     1.66     82.42                                    ______________________________________                                    

                  TABLE 18                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON FELT FORMATION TIME                                                        (seconds)                                                                     Variable        Effect  T-value    Probability                                ______________________________________                                        1.  Particle size    -8.75  0.58     38.58                                    2.  Dispersity      -13.75  0.91     57.23                                    3.  Mixture          -5.25  0.35     22.74                                    4.  Amount of pulp   -9.75  0.65     42.71                                    5.  Pulp freeness   -14.25  0.95     58.82                                    6.  Amount of particulate                                                                          -6.25  0.42     27.45                                    7.  Dummy           -24.75  1.64     82.18                                    ______________________________________                                    

                  TABLE 19                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON PERCENT ASH OF COMPOSITE                                                   Variable       Effect   T-value    Probability                                ______________________________________                                        1.  Particle size  -0.75    0.23     14.32                                    2.  Dispersity     -1.25    0.39     25.45                                    3.  Mixture        0.75     0.23     14.32                                    4.  Amount of pulp -5.75    1.78     84.96                                    5.  Pulp freeness  2.25     0.70     45.61                                    6.  Amount of particulate                                                                        -10.75   3.33     98.01                                    7.  Dummy          -6.25    1.93     87.64                                    ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON PRESSURE DROP                                                              (kg/cm.sup.2 at 6 ml/min)                                                     Variable        Effect  T-value    Probability                                ______________________________________                                        1.  Particle size   6.56    0.12     71.81                                    2.  Dispersity      -0.61   0.12     6.37                                     3.  Mixture         -6.91   1.34     74.02                                    4.  Amount of pulp  -2.89   0.56     37.14                                    5.  Pulp freeness   7.79    1.51     78.88                                    6.  Amount of particulate                                                                         -7.09   1.37     75.07                                    7.  Dummy           3.94    0.76     49.36                                    ______________________________________                                    

                  TABLE 21                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON RESOLUTION AT 6 ml/min                                                     Variable        Effect  T-value    Probability                                ______________________________________                                        1.  Particle size   0.15    0.30     19.22                                    2.  Dispersity      -0.28   0.57     38.13                                    3.  Mixture         0.92    1.88     86.67                                    4.  Amount of pulp  0.01    0.03     0.90                                     5.  Pulp freeness   -0.20   0.40     26.48                                    6.  Amount of particulate                                                                         -0.46   0.94     58.50                                    7.  Dummy           0.12    0.25     15.54                                    ______________________________________                                    

                  TABLE 22                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON H OF TOLUENE AT 0.2 ml/min                                                 Variable        Effect  T-value    Probability                                ______________________________________                                        1.  Particle size   -0.01   0.11     6.06                                     2.  Dispersity      0.14    1.15     67.33                                    3.  Mixture         -0.19   1.58     80.70                                    4.  Amount of pulp  -0.10   0.89     55.83                                    5.  Pulp freeness   0.11    0.94     58.68                                    6.  Amount of particulate                                                                         0.05    0.43     28.76                                    7.  Dummy           0.05    0.42     27.58                                    ______________________________________                                    

                  TABLE 23                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON H                                      OF TOLUENE AT 6 ml/min                                                        Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.30     0.81     51.75                                        2. Dispersity 0.42      1.12     66.27                                        3. Mixture    -0.52     1.40     75.98                                        4. Amount of pulp                                                                           -0.17     0.45     30.04                                        5. Pulp freeness                                                                            0.37      0.99     60.93                                        6. Amount of particulate                                                                    0.50      1.35     74.36                                        7. Dummy      0.13      0.36     23.54                                        ______________________________________                                    

                  TABLE 24                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON                                        HETP OF TOLUENE AT 12 ml/min                                                  Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.32     0.79     51.11                                        2. Dispersity 0.42      1.03     62.75                                        3. Mixture    -0.59     1.47     77.86                                        4. Amount of pulp                                                                           -0.23     0.58     38.51                                        5. Pulp freeness                                                                            0.39      0.98     60.15                                        6. Amount of particulate                                                                    0.50      1.24     70.74                                        7. Dummy      0.15      0.38     24.63                                        ______________________________________                                    

                  TABLE 25                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON H                                      OF DMP AT 0.2 ml/min                                                          Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.34     0.65     42.97                                        2. Dispersity 0.23      0.45     29.91                                        3. Mixture    -0.89     1.72     83.71                                        4. Amount of pulp                                                                           -0.43     0.84     53.42                                        5. Pulp freeness                                                                            0.01      0.03     0.89                                         6. Amount of particulate                                                                    0.31      0.59     39.38                                        7. Dummy      0.34      0.65     42.97                                        ______________________________________                                    

                  TABLE 26                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON H                                      DBP AT 0.2 ml/min                                                             Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.18     0.56     36.96                                        2. Dispersity 0.30      0.92     57.63                                        3. Mixture    -0.52     1.59     80.99                                        4. Amount of pulp                                                                           -0.13     0.40     26.37                                        5. Pulp freeness                                                                            0.24      0.72     47.16                                        6. Amount of particulate                                                                    0.25      0.75     48.62                                        7. Dummy      0.18      0.55     36.57                                        ______________________________________                                    

                  TABLE 27                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON H                                      DBP AT 6 ml/min                                                               Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.66     0.77     49.89                                        2. Dispersity 0.83      0.97     59.98                                        3. Mixture    -1.17     1.37     74.86                                        4. Amount of pulp                                                                           -0.59     0.69     45.02                                        5. Pulp freeness                                                                            0.73      0.85     54.30                                        6. Amount of particulate                                                                    0.79      0.93     57.89                                        7. Dummy      0.66      0.77     49.89                                        ______________________________________                                    

                  TABLE 28                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON k'                                     DBP AT 6 ml/min                                                               Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            0.02      0.19     11.43                                        2. Dispersity -0.18     1.94     87.79                                        3. Mixture    -0.03     0.30     19.32                                        4. Amount of pulp                                                                           -0.04     0.47     30.92                                        5. Pulp freeness                                                                            -0.15     1.67     82.75                                        6. Amount of particulate                                                                    -0.35     3.86     99.03                                        7. Dummy      -0.03     0.30     19.32                                        ______________________________________                                    

                  TABLE 29                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON                                        HETP × PRESSURE DROP AT 6 ml/min kg/cm.sup.2                            Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            7.53      0.44     29.38                                        2. Dispersity 11.27     0.66     43.67                                        3. Mixture    -27.55    1.62     81.67                                        4. Amount of pulp                                                                           -4.13     0.24     15.12                                        5. Pulp freeness                                                                            -6.93     0.41     26.94                                        6. Amount of particulate                                                                    -6.55     0.39     25.34                                        7. Dummy      14.53     0.86     54.32                                        ______________________________________                                    

                  TABLE 30                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON H                                      OF DBP AT 20 ml/min                                                           Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.27     0.53     35.13                                        2. Dispersity 0.42      0.81     52.09                                        3. Mixture    -0.88     1.71     83.48                                        4. Amount of pulp                                                                           -0.22     0.42     27.95                                        5. Pulp freeness                                                                            0.37      0.72     47.11                                        6. Amount of particulate                                                                    0.41      0.79     50.95                                        7. Dummy      0.20      0.39     25.70                                        ______________________________________                                    

                  TABLE 31                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES ON k'                                     OF DMP AT 20 ml/min                                                           Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            0.40      1.62     81.72                                        2. Dispersity -0.25     1.01     61.75                                        3. Mixture    0.14      0.58     38.63                                        4. Amount of pulp                                                                           0.01      0.03     1.15                                         5. Pulp freeness                                                                            -0.45     1.85     86.22                                        6. Amount of particulate                                                                    -0.93     3.81     98.96                                        7. Dummy      0.00      0.01     0.28                                         ______________________________________                                    

                  TABLE 32                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARAIBLES ON                                        H × PRESSURE DROP AT 20 ml/min                                          Variable      Effect      T-value  Probability                                ______________________________________                                        1. Particle size                                                                            358.50      0.50     33.36                                      2. Dispersity 402.50      0.56     37.41                                      3. Mixture    -1,203.50   1.68     83.04                                      4. Amount of pulp                                                                           -142.00     0.20     11.92                                      5. Pulp freeness                                                                            -480.50     0.67     44.19                                      6. Amount of particulate                                                                    -514.00     0.72     46.93                                      7. Dummy      551.00      0.77     49.85                                      ______________________________________                                    

                  TABLE 33                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES k'                                        OF DBP AT 20 ml/min                                                           Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            0.03      0.35     22.67                                        2. Dispersity -0.17     1.91     87.30                                        3. Mixture    -0.02     0.23     14.30                                        4. Amount of pulp                                                                           -0.10     1.16     67.83                                        5. Pulp freeness                                                                            -0.14     1.68     82.98                                        6. Amount of particulate                                                                    -0.28     3.30     97.95                                        7. Dummy      -0.04     0.41     26.78                                        ______________________________________                                    

                  TABLE 34                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           H OF DMP AT 20 ml/min                                                         Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            -0.32     0.66     43.72                                        2. Dispersity 0.45      0.92     57.64                                        3. Mixture    -0.76     1.56     80.23                                        4. Amount of pulp                                                                           -0.09     0.18     10.62                                        5. Pulp freeness                                                                            0.28      0.58     38.62                                        6. Amount of particulate                                                                    0.38      0.78     50.22                                        7. Dummy      0.25      0.52     34.61                                        ______________________________________                                    

                  TABLE 35                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON THE DBP RATIO OF k' AT 0.2 AND 20 ml/min                                   Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            0.20      1.96     73.97                                        2. Dispersity 0.46      4.41     85.41                                        3. Mixture    -0.15     1.45     69.23                                        4. Amount of pulp                                                                           0.35      3.36     81.83                                        5. Pulp freeness                                                                            -0.22     2.08     74.85                                        6. Amount of particulate                                                                    -0.01     0.11     31.02                                        7. Dummy      0.10      1.00     63.21                                        ______________________________________                                    

                  TABLE 36                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON THE DMP RATIO OF k' AT 0.2 AND 20 ml/min                                   Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            0.21      1.20     66.21                                        2. Dispersity 0.42      2.45     77.34                                        3. Mixture    -0.10     0.59     54.84                                        4. Amount of pulp                                                                           0.33      1.90     73.47                                        5. Pulp freeness                                                                            -0.14     0.80     59.54                                        6. Amount of particulate                                                                    0.05      0.30     44.67                                        7. Dummy      0.17      1.00     63.21                                        ______________________________________                                    

                  TABLE 37                                                        ______________________________________                                        EFFECT OF THE INDEPENDENT VARIABLES                                           ON THE RATIO OF PRESSURE DROP AT 20 and 6 ml/min                              Variable      Effect    T-value  Probability                                  ______________________________________                                        1. Particle size                                                                            0.08      8.50     92.34                                        2. Dispersity 0.09      8.50     92.34                                        3. Mixture    0.07      6.50     89.80                                        4. Amount of pulp                                                                           -0.02     2.00     74.27                                        5. Pulp freeness                                                                            0.07      6.50     89.80                                        6. Amount of particulate                                                                    -0.23     23.00    98.14                                        7. Dummy      0.01      1.00     63.21                                        ______________________________________                                    

                                      TABLE 38                                    __________________________________________________________________________    SUMMARY OF THE EFFECTS OF THE INDEPENDENT VARIABLES                                        Composite             Product of                                         Change                                                                             formation                                                                           Felt Pressure   pressure                                           in   weight                                                                              drainage                                                                           drop at                                                                            Reso- drop and                                                                            k' of                                                                            k' of                             Variable                                                                              variable                                                                           loss  time 6 ml/min                                                                           lution                                                                            H H     DBP                                                                              DMP                               __________________________________________________________________________    Particle size                                                                         -    +     +    +    0   - +     0  0                                 Partisil present                                                                           0     -    0    -   + +     -  0                                 Mixture present                                                                            0     0    -    +   - -     0  -                                 Amount of pulp                                                                        -    +     -    0    0   - 0     0  -                                 Pulp freeness                                                                         +    -     -    -    -   + -     -  0                                 Amount of                                                                             -    -     0    -    -   + -     -  -                                 particulate                                                                   __________________________________________________________________________     NOTE:                                                                         + = positive change.                                                          - =  negative change.                                                         0 = no change.                                                           

EXAMPLE 7

A column parameter which significantly influences effectiveness of acolumn to separate components is the amount of axial dispersion. It isconvenient to compare the dispersion obtained in different columns bymeans of a dispersion variance. When the peak eluting is Gaussian, twoparameters are needed to establish this curve--the mean and the standarddeviation. The variance of the dispersion curve is the square of thestandard deviation. The dimensionless variance, obtained by dividing thestandard deviation by the mean of the dispersion curve and then squaringthe result, can be used to compare the efficiency of various columns.

Following the procedure of Example 4, the dimensionless variances forthe chromatographic separation of DMP (dimethyl phthalate) and DBP(dibutyl phthalate) were obtained and are set forth in Table 39. Notethat the dispersion for the retained components was not substantiallygreater than the dispersion for the nonretained component toluene. Sincethe dispersion of the retained component depends upon the dispersion dueto back mixing in the column itself, any dispersion due to diffusionresistance, the variance due to diffusion or resistance can be obtainedby subtracting the toluene variance from the retained componentvariance. Such calculation is presented in Table 40. Note that most ofthe column variance is due to longitudinal diffusion or back mixing. Thecontribution of the diffusional resistance was approximately the samefor both DMP and DBP and these results are in contrast to the dispersionobtained with a particulate column of the Syloid 620 which is alsopresented in Table 40. Relatively sharp peaks were obtained with theunknown retained component toluene and substantially greater dispersionwas obtained on the retained components. This indicates that relativelylittle back mixing or axial diffusion is present in the silica column.There was significant differences in DBP, which is not as highlyretained as DMP. At the slower and faster flow rates examined, thediffusional resistance was greater in the particulate column than in thecomposite column. The contribution due to back mixing will varydepending on the particular composition of a composite column.

EXAMPLE 8

Following the procedure of Example 7, the pressure drops, H, retentionfactor k' and theoretical minimum column distance required for 99%separation of DMP and DBP were determined and are set forth in Table 40.The pressure drop is approximately linear with flow rate, indicatinglaminar flow conditions. The dispersion coefficient or amount of backmixing obtained is also apparently linear with flow rate. In general, Hfor the composite material did not vary as much with flow rate as theydid for a column containing 100% particulate. The insensitivity of thecomposite material with respect to flow rates is an advantage.

                                      TABLE 39                                    __________________________________________________________________________    Flow Rate                                                                           Velocity, V                                                                         Toluene                                                                            DBP  DMP  DBP  DMP                                           ml/min                                                                              cm/min                                                                              Variance                                                                           Variance                                                                           Variance                                                                           U/D.sub.eff L                                                                      U/D.sub.eff L                                 __________________________________________________________________________    Composite                                                                     0.2   0.06367                                                                             0.00102                                                                            0.001291                                                                           0.001684                                                                           0.000274                                                                           0.000667                                      0.5   0.159 0.00113                                                                            0.00157                                                                            0.00169                                                                            0.00044                                                                            0.000556                                      1     0.318 0.00129                                                                            0.00179                                                                            0.00172                                                                            0.000496                                                                           0.0004299                                     2     0.637 0.001414                                                                           0.00193                                                                            0.00194                                                                            0.0005118                                                                          0.000529                                      4     1.273 0.00146                                                                            0.002086                                                                           0.00210                                                                            0.000624                                                                           0.000643                                      6     1.91  0.00159                                                                            0.00222                                                                            0.00216                                                                            0.000654                                                                           0.0005977                                     8     2.55  0.00150                                                                            0.00233                                                                            0.00216                                                                            0.000832                                                                           0.000665                                      9.9   3.15  0.00158                                                                            0.00279                                                                            0.00211                                                                            0.00121                                                                            0.000535                                      Silica                                                                        1     0.318  --  0.00544                                                                            0.000603                                                                           0.00508                                                                            0.000311                                      2     0.637 0.000352                                                                            --   --   --   --                                           6     1.91  0.000419                                                                           0.00195                                                                            0.00133                                                                            0.00153                                                                            0.00091                                       9.9   3.15  0.000492                                                                           0.00224                                                                            0.00134                                                                            0.00175                                                                            0.000848                                      12.0  3.82  0.000571                                                                           0.00431                                                                            0.00167                                                                            0.00374                                                                            0.00110                                       __________________________________________________________________________

                                      TABLE 40                                    __________________________________________________________________________             Pressure                 Pressure                                    %   %    Drop               Separation                                                                          Drop           Total                        Syloid                                                                            250 CSF                                                                            (kg/cm.sup.2)                                                                      HETP (mm)                                                                            k'     Time  (kg/cm.sup.2)                                                                           Distance                                                                           Particulate                  620 Pulp (25 cm)                                                                            DBP                                                                              DMP DEP                                                                              DMP (min) (for 99% separation)                                                                    (cm) (gm)                         __________________________________________________________________________    30  10   35   3.014                                                                            2.972                                                                             1.1                                                                              3.0 4.00  29.3      20.2 0.90                         50  7.5  17.2 7.453                                                                            6.672                                                                             1.0                                                                              2.8 7.55  27.8      39   4.78                          0  5    26.3 -- --  -- --  1.17  13.4      9.87 0.66                         19  6    31.5 1.674                                                                            1.036                                                                             0.7                                                                              1.9 3.35  12.9      7.88 1.06                         44  6    37.8 1.85                                                                             1.343                                                                             2.9                                                                              6.6                                                   100 0    27.3 0.56                                                                             0.335                                                                              3.79                                                                            9.6 1.08  20.6      1.54  0.705                       __________________________________________________________________________

EXAMPLE 9

The column length needed to achieve a given separation depends on thetime of separation between the peaks and peak widths. The time betweenpeaks is a function of the capacity factor, k', and the width betweenpeaks can be expressed as the number of standard deviations (X). Thefollowing equation can be used to predict the column length necessary toobtain a specified separation

length=(experimental column length) (dimensionless variance) (X)² [sumof capacity factors plus two/difference between capacity factors]².

For the 70% Syloid 620 column described heretofore, 99% of the samplewas recovered and the number of standard deviations was 2.33.Accordingly, the column length required for separation of DMP and DBPwas only 7 cm. This equation does not take into account columnirregularities which may cause channeling and in turn cause peakbroadening. Elution curves are also not represented by a normaldistribution, particularly at high column loading, which tends tointroduce additional error. However, the equation is useful since itpermits ease of preliminary design and identification of significantvariables for design.

The capacity of a column is related to the time required for a componentto elute. Having a high capacity is not necessarily desirable sincelonger retention times actually lower production capacity if separationsare desired. Theoretically the capacity factor is equal to the productof the partition coefficient and the ratio of the exterior surface areaof the stationary phase to the volume of the mobile phase. Accordingly,the capacity factor of the stationary phase of the present invention canbe adjusted by changing the ratio of the surface area to the void volumeof the composite material. The void volume for all of the stationaryphases tested, as described in these examples was approximately constantand the fibers demonstrated no separation capacity for the componentstested. This suggests that the amount of particulate or particulatemixture in the composite is the major variable which can be used toinfluence the capacity factor of the composite material. A linearrelationship existed between the capacity factor and the ratio of theamount of silica to the void volume.

EXAMPLE 10 FRACTIONATION OF RECOVERED HUMAN PLASMA BY WEAK ANIONEXCHANGE MEDIA

A 10 ml aliquot of recovered human plasma diluted tenfold with 0.025 MpH 5.5 sodium acetate buffer (final pH 6.31) was passed through a columnconforming to the present invention measuring 12.5 cm high by 2.5 cmi.d. and packed with individual sorption elements containing 70% silicagel (70 micrometer average particle size modified by treatment with3-amino propyltriethoxy silane). Throughput rate was 5 ml/min. Thecolumn was sequentially eluted with

(1) 0.025 M sodium acetate buffer pH 6.31,

(2) 0.25 M sodium acetate buffer pH 5.5, 0.25 M sodium acetate buffer pH4.8 and

(3) 0.25 M sodium acetate buffer pH 4.0.

The elution profiles of the four fractions are set forth in FIG. 11.Fractions of approximately 20 ml were collected and twelve were selectedto assay for albumin (Sigma No. 630) and globulins (Sigma No. 560). Theelution profiles of the twelve fractions are given below in Table 41 andthe characteristics of six pooled fractions are given in Table 42.

                                      TABLE 41                                    __________________________________________________________________________    Elution Profiles of Eleven Selected Fractions                                               Albumen*                                                                            Globulin**     Relative                                   Tube Number                                                                          O.D. 280 nm                                                                          (O.D.630)                                                                           (O.D.560)                                                                           Albumen/globulin                                                                       Ratio.sup.+                                __________________________________________________________________________    Initial       0.377 0.185 2.03     1                                           4     0.45   0     0.026 0        0                                           7     1.22   0     0.051 0        0                                          11     0.27   0     0.004 0        0                                          18     1.60   0.17  0.029 0.59     0.29                                       20     0.90   0.054 0.040 1.35     0.67                                       22     0.75   0.026 0.019 4.52     2.23                                       24     0.66   0.103 0.010 10.30    5.07                                       30     0.41   0.080 0.006 13.33    6.57                                       38     >2     0.232 0.035 6.62     3.26                                       40     0.96   0.102 0.021 4.84     2.39                                       48     2.00   0.052 0.051 1.01     0.50                                       49     1.40   0.039 0.044 0.89     0.44                                       __________________________________________________________________________     *Sigma No. 630 Assay                                                          **Sigma No. 560 Assay                                                         .sup.+ Albumen/globulin ratio of Tube divided by ratio of initial sample.

                  TABLE 42                                                        ______________________________________                                        Characteristics of Six Fooled Fractions                                       Fraction                                                                             Tubes     Volume (ml)                                                                              Buffer* pH                                                                              NaCl (m)                                ______________________________________                                        I**     1-11     36         5.5       0                                       II     12-20     30         4.3       0                                       III    21-32     40         4.3       0.2                                     IV     33-50     61         4.0       0.2                                     V      51-63     44         3.8       0.2                                     VI     64-80     57         3.8       0.5                                     ______________________________________                                         *Buffer = 0.025 M sodium acetate                                              **Sample application during this fraction                                

EXAMPLE 11 GLUCOAMYLASE PURIFICATION BY WEAK ANION EXCHANGE MEDIA

A commercial glucoamylase solution was dialyzed against 0.025 M sodiumacetate buffer pH 5.5 and filtered through 50 S Zeta Plus cationallymodified filter media (AMF Incorporated, White Plains, New York). A 1 mlsample of the solution (45.6 mg/ml protein) was then applied to achromatographic column (10 cm high and 0.9 cm i.d.) at 1 ml/min (22 PSI)at room temperature. The column was packed with sorption elements of thetype described in Example 10. A total of 80 tubes of 3.3 ml fractionswere collected and pooled into six fractions corresponding to thedifferent pH and ionic strength step changes in the elution of theproteins (Table 43). The assay results of the six fractions compared tothe initial protein sample are given in Table 43 below. These data showthat ˜77% of the initial activity is recovered in Fraction VI with a 1.8fold purification.

                  TABLE 43                                                        ______________________________________                                        Protein and Enzyme Activity Results                                                     Protein Enzyme             Specific                                 Sample    (mg)*   Activity**                                                                              Activity/mg                                                                            Activity.sup.+                           ______________________________________                                        Starting  45.6    7.30      0.16     1                                        Fraction I                                                                              1.1     0         0        0                                        Fraction II                                                                             0.6     0         0        0                                        Fraction III                                                                            1.0     0         0        0                                        Fraction IV                                                                             11.6    0.61      0.053    0.33                                     Fraction V                                                                              6.6     0.68      0.13     0.80                                     Fraction VI                                                                             17.8    4.88      0.28     1.80                                     Fraction  38.1    6.33      0.17                                              Totals                                                                        ______________________________________                                         *Lowry assay                                                                  **Coupled assay to measure starch hydrolysis to glucose                       .sup.+ Relative to Starting activity per mg                              

EXAMPLE 12 PURIFICATION OF IMMUNOGLOBULIN G (Ig G)

Each of four chromatographic columns 10 cm high×10 mm i.d. packed with0.2 g of the sorption element media described in Example 10 wereoperated at a flow rate of 1.5 ml/min. The Ig G samples (ammoniumsulfate fractionation of human serum obtained from Immunoreagents Inc.,Sequin, Tex.) were dialyzed against 0.05 M sodium phosphate, pH 6.8before passage through the column. The columns were eluted with 0.05 Msodium phosphate pH 6.8 and 0.025 M sodium acetate with 0.5 M sodiumchloride, pH 4.0.

The results obtained with each column are set forth in the followingTable:

                                      TABLE 44                                    __________________________________________________________________________    Results of Separation                                                                                                 % OF TOTAL                             COLUMN                                                                              SAMPLE                                                                             6.8 RECOVERY                                                                         4.0 RECOVERY                                                                         TOTAL RECOVERY                                                                       % RECOVERY                                                                           % RECOVERY IN 6.8                                                                       ##STR1##                    __________________________________________________________________________    1     15.5mg                                                                               9.13mg                                                                               2.74mg                                                                              12.915mg                                                                             83%    70.69%   3.33                         2     32mg  21.08mg                                                                               6.95mg                                                                              29.66mg                                                                              93.9%  71.06%   3.03                         3     49.8mg                                                                              33.09mg                                                                              10.7mg 45.99mg                                                                              92.3%  71.96%   3.092                        4     65.48mg                                                                             48.41mg                                                                              21.38mg                                                                              62.68mg                                                                              95.7%  77.23%   3.9                          __________________________________________________________________________

From Table 44 it can be seen that the first three columns yieldedsimilar results. If it is assumed that the separation in the smallestsample was the optimum, it is seen that the distribution of protein(IgG) in the next two samples is very similar with approximately 71% ofthe recoverable protein eluting with the 6.8 buffer. In the fourthsample, the protein in the 6.8 fraction increased to 77.2% of therecoverable protein, indicating a probable overloading of the column.From this information, the maximum capacity is found to be 25 mg pergram of media.

In order to qualify the purity of the pH 6.8 elution sample, equalamounts of IgG eluant from the 49.8 mg column and the 65.48 mg columnwere re-chromatographed. Equal amounts from these 6.8 elutants (3B and4B) were re-chromatographed (3C and 4C). The results of the separationare set forth in the following table:

                                      TABLE 45                                    __________________________________________________________________________    Results of Separation of Re-chromatographed Eluants                            SAMPLE                                                                              Mg APPLIED                                                                           6.8 ELUTION                                                                         4.0 ELUTION                                                                         TOTAL RECOVERY                                                                       % TOTAL RECOVERY IN 6.8                                                                     ##STR2##                       __________________________________________________________________________    3A    49.8mg  33.09mg                                                                             10.7mg                                                                              45.99mg                                                                              71%          3.09                            4A    65.48mg 48.41mg                                                                             12.38mg                                                                             62.68mg                                                                              77.23%       3.9                             3B    16mg    12.47mg                                                                              .779mg                                                                             13.866mg                                                                             89%          16.01                           4B    16.1mg  12.61mg                                                                              1.373mg                                                                            14.617mg                                                                             86.3%        9.18                            3C     5.79mg  4.61mg                                                                              .126mg                                                                              4.85mg                                                                              94%          36.58                           4C     5.8mg   4.52mg                                                                              .138mg                                                                              4.78mg                                                                              94%          32.75                           __________________________________________________________________________

It can be seen from the elution profiles of 3B and 4B that the 6.8elution of 3A was purer than that from 4A. In the B series, similarapplications yielded different ratios of protein in the 6.8 vs. 4.0elutions. When the purified IgG samples from the B series werere-chromatographed the elution profiles were almost identical.

EXAMPLE 13 PREPARATIVE SCALE IMMUNOGLOBULIN G (IgG) PURIFICATION

A chromatographic column according to the invention herein (24 cmhigh×2.5 cm i.d. packed with 33 g of individual sorption elements asdescribed in Example 10) was equilibrated as follows:

500 mls water

200 mls 0.1 M pH 6.8 sodium phosphate

800 mls 0.025 pH 4.0 sodium acetate with 0.5 M sodium chloride

500 mls. 0.1 M pH 6.8 sodium phosphate

1500 mls. 0.05 M pH 6.8 sodium phosphate

Equilibration was carried out at 10 mls per minute. The void volume ofthe column was approximately 80 mls.

An IgG sample as described in Example 12 was dialyzed against 0.05 M pH6.8 sodium phosphate before being applied to the column. After dialysis,a 1:100 dilution had an OD 280-0.258 which is equal to 19.1 mg/ml (ODIgG, 1 mg/ml=1.35). 43 mls (821.3 mg) of the IgG solution were passedthrough the column. Elution was carried out with 0.05 M pH 6.8 sodiumphosphate followed by 0.025 M pH 4.0 sodium acetate with 0.5 M sodiumchloride. Flow rate was 4 ml per minute. The results of the separationare set forth below and the elution characteristics of the 6.8 and 4.0recoveries are graphically represented in FIG. 12.

Sample--821.3 mg

6.8 recovery--535.28 mg

4.0 recovery--179.51 mg

Total recovery--724.43 mg

% total recovery--88.48%

% of total in 6.8--73.68%

6.8/4.0--2.98

EXAMPLE 14

In order to compare a column containing the solid stationary phase ofthis invention with certain prior art columns, in particular U.S. Pat.No. 3,455,818 to Leifield and U.S. Pat. No. 3,856,681 to Huber, whichsuggest the use of a plurality of sheets of chromatographic mediaarranged adjacent to each other with the thickness dimension of thelayers extending substantially perpendicular to the primary fluid-flowaccess, a "square" molecular separation column was constructed. Thecolumn consisted of a trough 25 millimeters wide, 25 millimeters deep,and 259 millimeters long. A cover was provided which bolted to thetrough with an "O" ring to seal the joint. A 0.16 cm hole was drilledinto the center of the ends for entrance and exit of the mobile phase. A0.16 cm thick 25 millimeter square stainless steel frit of 2 μm porositydistributed the solvent at both ends.

The column was packed with sheets consisting of 24% unrefined pulp, 6%of -250 highly refined pulp, and 70% Syloid 620. The material was feltedinto 30.50 circular sheets. For the vertical packing (prior art) six toeight 25×250 millimeter strips were cut. These were then placed in thecolumn and the cover bolted on. The media was then tested for molecularseparation performance. For the horizontal packing, i.e. thesubstantially homogeneous solid stationary phase of this invention, thesame procedure was followed using 25×25 millimeter squares-stackedhorizontally along the trough. Both the horizontal strips (invention)and the vertical strips (prior art) were tested in duplicate using aVarian Model 5000 Liquid Chromatograph. In an additional test, both thevertical and horizontal packing were packed as before and then anadditional pad or strip added under high pressure to ensure a highpacking density in the column.

Samples containing: (1) benzene, (2) beta naphthol, (3)para-nitro-phenol, (4) benzoic acid, and (5) dimethyl phthalate wereevaluated. The mobile phase was hexane. The ultraviolet detector usedwas set at 254 nanometers.

These tests showed that the horizontal packing of the invention gaveabout an 8 fold increase in column efficiency and much sharper peakshapes indicating much better chromatographic performance than thevertical strips (prior art). This was in spite of the fact that thecolumn was designed and the flow geometry optimized specifically for thevertical strips (prior art). Generally, all of the chromatograms fromthe injection of the above listed samples showed very "broad" peakshapes and very "bad" tailing for the vertical packing (prior art) whencompared to the horizontal packing (invention).

Various changes and modifications can be made in the process andproducts of this invention without departing from the spirit and scopethereof. The various embodiments which have been described herein werefor the purpose of further illustrating the invention, but were notintended to limit it.

We claim:
 1. A chromatography column for effecting differentialdistribution, between two phases, of the components of a sample flowingtherethrough, said column containing a solid stationary phasesubstantially homogeneous with respect to each component thereof, whichcomprises a porous matrix of fiber having particulate immobilizedtherein, at least one of said fiber or particulate therein havingchromatographic functionality and being effective for chromatographicseparation, wherein said solid stationary phase comprises a plurality ofsheets of said solid stationary phase elements, the edges of saidelements cooperating with the interior wall of the column to form asubstantially fluid tight seal therewith, preventing any appreciableskewing or by-pass of fluid around the edges of the elements.
 2. Thechromatography column of claim 1, wherein the particulate haschromatographic functionality and is effective for chromatographicseparation.
 3. The chromatography column of claim 2, wherein the fibersare cellulose.
 4. The chromatography column of claim 3, wherein thematrix comprises a major amount of long self bonding structural fiberand a minor amount of refined pulp fiber whose Canadian StandardFreeness is in the range of about +100 to -600 ml.
 5. The chromatographycolumn of claim 4, wherein the ratio of structural fiber to refined pulpfiber is 2:1 to 10:1.
 6. The chromatography column of claim 5, whereinthe ratio is 3:1 to 5:1.
 7. The chromatography column of claims 1, 2, 3,4, 5 or 6, wherein the amount of particulate is at least 10 weightpercent of said solid stationary phase.
 8. The chromatography column ofclaim 7, wherein the amount of particulate is from 10 to 80 weightpercent of said solid stationary phase.
 9. The chromatography column ofclaims 1, 2, 3, 4, 5 or 6, wherein said particulate has a mean particlesize of about 5-100 microns.
 10. The chromatography column of claim 4,wherein said structural fiber is cellulose having a Canadian StandardFreeness of +400 to +800 ml.
 11. The chromatography column of claim 1,2, 3, 4, 5 or 6, wherein said column is cylindrical and said sheets arediscs.
 12. The chromatography column of claim 11, wherein said solidstationary phase is hydrophilic swellable.
 13. The chromatography columnof claim 12, wherein said elements form a fluid-tight seal with theinterior wall of the cylinder by water swellable fit therewith.
 14. Thechromatography column of claim 13, wherein said particulate has a meanparticle size of from about 5 to 100 microns and is about 10 to 80weight percent of said solid stationary phase, said structural fiber iscellulose having a Canadian Standard Freeness of +400 to +800 ml and aratio of said structural fiber and refined pulp fiber is 3:1-5:1. 15.The chromatography column of claim 1, 2, 3 or 4, wherein said elementsform a fluid-tight seal with the interior wall of the cylinder bycompression friction fit therewith.
 16. In a method of effecting achromatographic separation by effecting a differential distribution of asample's components between two phases by passing a mobile phase througha chromatography column containing a solid stationary phase, theimprovement which comprises employing as said stationary phase a porousmatrix of fiber having particulate immobilized therein, at least one ofsaid fiber or particulate having chromatographic functionality and beingeffective for chromatographic separation, said solid stationary phasebeing substantially homogeneous with respect to each component thereof,wherein said solid stationary phase comprises a plurality of sheets ofsaid solid stationary phase elements, the edges of said elementscooperating with the interior wall of the column to form a substantiallyfluid tight seal therewith, preventing any appreciable skewing or bypassof fluid around the edges of the elements.
 17. The method claim 16,wherein the particulate has chromatographic functionality and iseffective for chromatographic separation.
 18. The method of claim 16,wherein the mobile phase is liquid.
 19. The method of claim 17, whereinthe fibers are cellulose.
 20. The method of claim 19, wherein the matrixcomprises a major amount of long self bonding structural fiber and aminor amount of refined pulp fiber whose Canadian Standard Freeness isin the range of about +100 to -600 ml.
 21. The method of claim 20,wherein the ratio of structural fiber to refined pulp fiber is 2:1 to10:1.
 22. The method of claim 21, wherein the ratio is 3:1 to 5:1. 23.The method of claims 16, 17, 18, 19 or 20 wherein the amount ofparticulate is at least 10 percent of said solid stationary phase. 24.The method of claim 23, wherein the amount of particulate is from 10 to80 weight percent of said solid stationary phase.
 25. The method ofclaims 16, 17, 18, 19 or 20, wherein said particulate has a meanparticle size of about 5 to 100 microns.
 26. The method of claim 20,wherein said structural fiber is cellulose having a Canadian StandardFreeness of +400 to +800 ml.
 27. The method of claim 16, 17, 18, 19, 20,21 or 22, wherein said column is cylindrical and said sheets are discs.28. The method of claim 27, wherein said solid stationary phase ishydrophilic swellable.
 29. The method of claim 28, wherein said elementsform a fluid-tight seal with the interior wall of the cylinder by waterswellable fit therewith.
 30. The method of claim 29 wherein saidparticulate has a mean particle size of from about 5 to 100 microns andis about 10 to 80 weight percent of said solid stationary phase, saidstructural fiber is cellulose having a Canadian Standard Freeness of+400 to +800 ml and the ratio of said structural fiber and refined pulpfiber is 3:1-5:1.